By watching the rate and positioning of gene transcription in real time, researchers get a closer look at how embryos establish gene expression patterns. Learn more...

A team of scientists has adapted techniques originally designed for use in
single-celled organisms to track real-time transcription in developing Drosophila
embryos. Their measurements have already

The anterior region of the developing fly embryo had many more actively transcribing nuclei than the center of the embryo

provided new insight into how embryos establish spatial transcription profiles
that are key to the body plan of the developing organism. “With this method,
we’ve brought the embryo up to the task of answering basic quantitative
questions about transcription,” said Thomas Gregor of Princeton University,
senior author of a new paper in the journal Current Biology (1).

Early in development, embryos establish gradients that give the growing mass
of cells a sense of direction. One gene might be expressed at higher levels
on one side of the embryo, while another has the reverse pattern of
expression. Such patterns are vital for ensuring the correct differentiation
of the cells forming the body.

At a macroscopic level, these gradients are well-established. But how they are
set up at a molecular level has been less clear. Many hypotheses revolve
around transcription thresholds where a cell switches from one state to
another when it reaches a particular level of gene expression. Other
theories involve bursts of transcription that vary throughout the cell
cycle, rather than smooth increases and decreases.

Gregor’s team wanted to get more information on exactly how and when such gene
expression patterns appear and how the boundary between the “on” and “off”
states of a gene is established. So they turned to a method that uses
fluorescence to light up newly synthesized mRNA transcripts. The gene of
interest is engineered so that its RNA transcript forms stem loops during
transcription. These loops are then bound by specially designed molecules
fused to green fluorescent protein (GFP), which can be visualized with
microscopy.

The scientists used the approach to quantify the rate of transcription of the
developmentally relevant gene hunchback in growing Drosophila
embryos. “With that initial data on rate of transcription and timing of
transcription, we could come up with a model that partially explained the
transcription boundary we saw,” said Gregor. “But it was still off by a
factor of two.”

The team noticed that transcription was either on or off; it wasn’t a matter
of reaching a threshold.“The nuclei can adopt an active or an inactive state
that is basically random,” said Gregor. “And in the transition region is
where we saw nuclei randomly adopting these different states.” When the
scientists added this new observation into their model, it fully explained
the transcription boundary.

Earlier this year, Gregor’s lab optimized a different fluorescence method—a
variation of fluorescence in situ hybridization (FISH)—to count individual
mRNA transcripts in an embryo (2). By combining the two approaches, his team
plans to move towards a fully quantitative picture of transcription and its
dynamic regulation in living embryos.

“What these two methods have brought us is that we can now use the embryo to
study transcription in the way that other scientists use single-celled
organisms, but exploiting all the advantages that come with multicellular
organisms,” said Gregor.